The invention relates to the diagnosis and treatment of conditions associated with serotonin-mediated cellular responses.
The biogenic amine serotonin plays a role in the modulation of neuronal synaptic events as well as non-neuronal cellular signaling. Serotonin acts by binding to receptors on a variety of cells. These receptors fall into two broad functional and structural categories, those acting through G-proteins to mediate intracellular signaling, and those that form ion channels. It is generally believed that serotonim may act by binding to either G-protein-coupled seven-pass transmembrane receptors, or serotonin-gated cation channels. There are six major classes of G-protein-coupled receptors, each with numerous subtypes. Thus far, there is only one class of serotonin-gated ion channels, the 5-HT3 receptor. G-protein-coupled responses can be either excitatory or inhibitory upon activation by serotonin. Activation of G-protein-coupled receptors by serotonin generally mediates responses which are slower-acting and longer-lasting, while ion channels mediate fast-acting and transitory responses. The 5-HT3 receptor, comprised of the conducting subunit, 5-HT3a and a regulatory subunit, 5-HT3b appears to exclusively elicit excitatory responses that are generally fast-acting and transitory.
The 5-HT3 receptor is selectively permeable to cations only, such as Na+ or K+, and is very slightly permeable to Ca2+. The influx of cations, such as Na+ into a cell results in depolarization and excitatory neurotransmission. Efflux of cations, such as K+ hyperpolarizes the cell, thereby reducing the likelihood of excitation, and generally leads to inhibitory neurotransmission. Since the resting membrane of a typical cell/neuron is much less permeable to Na+ influx than to K+ efflux, the opening of a non-selective Na+/K+ channel, such as the 5-HT3 receptor leads to a dramatic influx of Na+, leading to depolarization and excitation.
Serotonin has been implicated in the etiology of many disease states, including depression, panic disorders, obsessive compulsive disorder, cardiac abnormalities, sleep disorders, eating disorders, nausea and vomiting, gastrointestinal cramps, and migraines. G-protein-coupled serotonin receptors have been implicated in the control of mood (5-HT1A), migraine (5-HT1B), pain perception (5-HT1D), smooth muscle contraction (5-HT2A, 5-HT7), anxiety (5-HT2B), and nausea (5-HT4). Activation of the 5-HT3 receptor by serotonin can either stimulate or inhibit cardiac function, induce vasodilation, affect lung and intestinal function, cause pain and sensitization of nociceptive neurons, and induce nausea and vomiting. Not surprisingly, many treatments for these disorders are thought to act through serotonergic pathways.
Several classes of drugs thought to modulate the serotonergic pathway exist. For example, selective serotonin re-uptake inhibitors (SSRIs) are used to treat depression. These antidepressants, including Prozac, Zoloft, and Paxil, are believed to act by potentiating serotonin levels at the synapse. Drugs, such as Imitrex, used to treat migraine headaches, act as selective serotonin receptor agonists. Other groups of drugs used to affect mood include monoamine oxidase inhibitors, and selective serotonin receptor antagonists.
While these drugs are administered to humans to treat the above-described disease states, the patients often unpredictably experience a number of side-effects including insomnia, anxiety, chest pain, hypertension, nausea, anorexia, sweating, chills, vomiting, diarrhea, constipation, decreased libido, and abnormal ejaculation. It has been hypothesized that the side-effects result from multiple receptor activation or inactivation when a serotonin agonist or antagonist is given as a treatment. Some of these improperly activated or inactivated receptors may lead to fast- or slow- acting excitatory responses, or slow-acting inhibitory responses, when really only one specific type of response is desired.
A better understanding of serotonin-associated cellular communication could greatly facilitate the discovery of drugs and therapeutic methodologies to treat a broad range of conditions with fewer of the serious and variable side-effects prevalent with currently available drugs that interface with the serotonin pathway. Exactly how the currently available drugs that interface with the serotonin pathway work is not well understood. Agonists, antagonists, and especially serotonin re-uptake inhibitors could affect numerous serotonin receptor subtypes, and the final outcome may be a combined readout of all these varied, and sometimes antagonistic, pathways. It has been hypothesized that the various undesirable side-effects of a given drug""s action is the result of such unwanted activation of the serotonin pathways that are not specific to the condition being treated. Therefore, compounds with a greater specificity for a specific serotonin receptor, for a limited subset of serotonin receptors, or for a specific subtype of a particular class of serotonin receptors would be invaluable to the field of therapeutics for serotonin-mediated disease states.
We have discovered a serotonin-gated ion channel, MOD-1, that is exclusively permeable to chloride ions, and is not permeable to sodium, potassium, or calcium ions. Activation of this anion channel is most likely to result in an inhibitory response. In some circumstances that are dependent on the reversal potential for chloride (which is a function of the concentration of chloride inside and outside the cell) activation of anion channels could result in an excitatory response. Therefore, it is conceivable that activation of a serotonin-gated anion channel could also result in excitatory neurotransmission.
With the discovery of MOD-1 and the serotonin-gated anion channel that it forms, comes the realization that serotonin may mediate fast-acting, and transitory, inhibitory responses in addition to excitatory responses. It is possible that the activation/inactivation of a MOD-1-like serotonin-gated anion channel, in humans, is associated with some of the effects and/or side-effects of existing serotonin-based drugs. It is also conceivable that many of the serotonin-related diseases are exclusively due to defects in, or associated with, a serotonin-gated anion channel. None of the currently available drugs have been designed to effectively and specifically target this receptor. Therefore, the discoveries of a serotonin-gated anion channel and the gene that encodes it are invaluable tools for use in discovering diagnostic and therapeutic compounds for the detection and treatment of conditions associated with serotonin-mediated cellular responses.
One way in which a serotonin-gated anion channel can be used as a tool in drug discovery is by screening existing drugs or drug candidates for their effects on serotonin-gated anion channel activity. Such an experiment can be done using MOD-1 or other serotonin-gated anion channels from non-mammals, such as nematodes, or from lower mammals or humans. Understanding how drugs affect, or do not affect, this anion channel will lend better insight into the overall effect of a drug""s mechanism of action. Also, a better understanding of how this serotonin-gated anion channel is regulated will contribute to a better understanding of how current therapies work. Furthermore, information gained from this screen will permit the development of drugs with higher specificities for a particular type of serotonin-binding receptor that will mediate only the desired response. Such drugs include those which do not activate a serotonin-gated anion channel, but do activate other serotonin receptors; those that act specifically on a serotonin-gated anion channel, but not on other serotonin receptors; or those that activate a subset of the various serotonin receptors.
Methods of drug discovery are not limited to screening available drugs only, but also to all compounds and their derivatives that were extracted or synthesized during the development of a given drug affecting serotonin-mediated cellular processes. Furthermore, a de novo screen of chemicals can be conducted, with no bias regarding possible functional relevance, for effects on this class of serotonin-gated anion channels.
In a first aspect, the invention features a substantially pure polypeptide that is a serotonin-gated anion channel. In one embodiment, the polypeptide is a substantially pure serotonin-gated anion channel that is permeable to chloride ions. In another embodiment, the polypeptide is MOD-1. In a further embodiment, the polypeptide is a subunit that makes up a multi-subunit serotonin-gated anion channel, permeable to chloride ions. Preferably this polypeptide is from Caenorhabditis elegans (C. elegans). More preferably this polypeptide is mammalian. Most preferably this polypeptide is human.
In still another embodiment of the invention, the serotonin-gated anion channel is activated by a lower concentration of serotonin than that required to activate the 5-HT3 receptor. For example, the serotonin-gated anion channel may have a higher binding affinity for serotonin than the 5-HT3 receptor. This higher affinity can be assessed by calculating and comparing the dissociation constants of serotonin binding to the 5-HT3 receptor and to a serotonin-gated anion channel.
In another aspect, the invention features a substantially pure nucleic acid sequence encoding a serotonin-gated anion channel. In one embodiment, the substantially pure nucleic acid sequence encodes a serotonin-gated anion channel that is permeable to chloride ions. In another embodiment, the nucleic acid sequence is mod-1, and encodes the MOD-1 polypeptide. Preferably this nucleic acid sequence is from C. elegans. More preferably the nucleic acid sequence is mammalian. Most preferably the nucleic acid sequence is human.
In another aspect, the invention features an antibody that preferably specifically binds to a serotonin-gated anion channel. In one embodiment, the antibody binds to a serotonin-gated anion channel that is permeable to chloride ions. This invention includes polyclonal, as well as monoclonal antibodies to the serotonin-gated anion channel.
In another aspect, the invention features a C. elegans strain having a mutant clod-1 gene. In one embodiment of the invention, the C. elegans strain has a mutant mod-1 gene that does not function as a chloride channel. In another embodiment, the strain has a mutant mod-1 gene that acts in a dominant-negative manner. In another embodiment, the strain has a mutant mod-1 gene that encodes a polypeptide that is constitutively active.
In another aspect, the invention features a method for identifying a compound that modulates the biological activity of a serotonin-gated anion channel. The method includes the steps of: (a) administering a test compound to a serotonin-gated anion channel, and (b) assaying a modulation in the biological activity of a serotonin-gated anion channel. Assaying the modulation of biological activity may be done by measuring the current carried through a channel, or by measuring the amount of serotonin binding to a serotonin-gated anion channel. The assay can also be a bioassay that involves measuring the rate of locomotion in nematodes having a serotonin-gated anion channel. In one embodiment, the serotonin-gated anion channel is from nematodes. In another embodiment, the serotonin-gated anion channel is from a rat, mouse, or human, and is inserted into a C. elegans that either has or does not have a wild-type C. elegans serotonin-gated anion channel. In another embodiment, the serotonin-gated anion channel is a chimeric molecule between the serotonin-gated anion channels from various species. In further various embodiments of this aspect, the channel is in a cell, for example, a neuron or a non-neuronal cell. The channel may also be in a lipid bi-layer, a mammal, or a nematode. In yet another embodiment, the serotonin-gated anion channel comprises sufficient MOD-1 protein to form a serotonin-gated anion channel.
In the above aspect of the invention, the test compound is administered in the absence or presence of serotonin, and is administered prior to, simultaneously with, or after administration of serotonin. Administration may also be in the absence or presence of known drugs that interface with the serotonin pathway. The modulation of the biological activity may be either agonistic or antagonistic. The compound may also be a cell lysate, or isolated from a cell lysate, and may be administered prior to, simultaneously with, or after administration of serotonin, or any known effector of serotonin-mediated cellular processes.
In another aspect, the invention features a method for treating a condition in a patient by administering an agonist or antagonist of a serotonin-gated anion channel to the patient. Conditions that are treated include migraine headaches, loss of appetite, gain of appetite, insomnia, inability to wake up, memory loss, inability to learn, nausea and vomiting, gastrointestinal cramps, body temperature deregulation, moods, including depression or mania, abnormal sexual or hallucinogenic behavior, abnormal cardiovascular function, abnormal muscle contraction, and abnormal endocrine regulation.
In another aspect, the invention features a diagnostic probe for measurement of a serotonin-gated anion channel, either wild-type or mutant, for detecting conditions associated with serotonin-mediated cellular responses. Measurement of a serotonin-gated anion channel includes, but is not limited to detection of nucleic acid levels that code for a serotonin-gated anion channel, levels of a polypeptide that can function as a serotonin-gated anion channel, single strand confirmation polymorphism analyses, or the flow of current across a membrane. In one embodiment, the probe is a nucleic acid sequence that encodes a serotonin-gated anion channel, for example, MOD-1, or a polypeptide that is a serotonin-gated anion channel, or an antibody that binds to a serotonin-gated anion channel. In yet another embodiment, the probe is standard electrophysiology voltage clamping equipment that measure the activity of a serotonin-gated anion channel. In still another embodiment, the diagnostic probe is used for pharmacogenetics, i.e., in the analyses of conditions associated with serotonin-mediated cellular responses within an individual, family, or families.
In another aspect, the invention features a method for characterizing drugs associated with serotonin-mediated cellular responses, by measuring serotonin-gated anion channel activity upon drug exposure. The drugs include those already currently available for the treatment of serotonin-mediated responses, as well as small molecules that are similar in structure to these drugs. This invention also includes the discovery of any compounds which have not yet been identified as therapeutic for serotonin-mediated cellular responses.
In another aspect, the invention features a method for decreasing serotonin-gated anion channel function by decreasing the level of a serotonin-gated anion channel polypeptide with antisense RNA to the serotonin-gated anion channel, and the antisense RNA itself. Preferably the level of the serotonin-gated anion channel is deceased at least 25%, more preferably at least 50%, 70%, or 80%, and most preferably at least 95%, compared to a control (e.g., a serotonin-gated anion channel that is not contacted with an antisense RNA, or that is contacted with a nonsense RNA sequence). In addition, preferably the antisense RNA is antisense mod-1 RNA. Such nucleic acids of the invention and methods for using them may be identified according to a method involving: (a) providing a cell sample; (b) introducing by transformation into tile cell sample, a test nucleic acid sequence for a serotonin-gated anion channel; (c) expressing the test nucleic acid of a serotonin-gated anion channel within the cell sample: and (d) determining whether the cell sample exhibits altered serotonin-gated anion channel activity, whereby either increased or decreased channel activity identifies a nucleic acid which may be used to alter serotonin-gated anion channel function. Preferably the cell is a non-neuronal cell. Most preferably the cell is a neuronal cell.
In another aspect, the invention features a method for decreasing the function of a serotonin-gated anion channel by administering an antibody that specifically binds to a serotonin-gated anion channel, or binds to a peptide from that channel. This method includes, but is not limited to, using an antibody to MOD-1 as the antibody, and a channel formed by any MOD-1 polypeptide as the channel whose function is inhibited. The methods also includes using a mammalian antibody to decrease the function of a mammalian serotonin-gated anion channel. This method also includes administration of the antibody in vivo or in vitro.
In another aspect, the invention features a method for modulating serotonin-gated anion channel activity using a nucleic acid vector encoding a serotonin-gated anion channel, and administering enough vector to alter activity of the serotonin-gated anion channels of at least one cell. In one embodiment, the vector is operably linked to a promoter. In other embodiments, the vector encodes a wild-type serotonin-gated anion channel or a mutant serotonin-gated anion channel. The mutant serotonin-gated anion channel may include, but is not limited to, a mutant channel that is a loss-of-function mutant, a dominant negative mutant, or a constitutively active mutant. In a preferred embodiment, the nucleic acid vector encodes a wild-type or mutant MOD-1 polypeptide. In other embodiments, administration may occur in vitro or in vivo. In further embodiments, the vector encodes a polypeptide that affects the function of a serotonin-gated anion channel, or that affects molecules that are targeted subsequent to activation of a serotonin-gated anion channel. These molecules include, but are not limited to, protein kinases, protein phosphatases, and proteases.
In another aspect, the invention features a method for testing a patient having a serotonin-mediated condition, for his/her pre-disposition to respond to therapy, or to experience side-effects due to administration of a specific therapy. The method comprises:
(a) determining the characteristics of a serotonin-gated anion channel from tissues of the patient, where the characteristics are indicative of the abnormal activity of a serotonin-gated anion channel; and
(b) administering to the patient a suitable therapeutic agent relative to the degree of abnormal activity of the serotonin-gated anion channel in step (a).
In various embodiments, the abnormal activity is due to mutations in a serotonin-gated anion channel protein, altered levels of synthesis of mRNA of a serotonin-gated anion channel, altered serotonin-gated anion channel protein levels in tissues. In another embodiment, the method further comprises characterizing other serotonin-mediated receptors in the patients. Therapeutic agents to be used in accordance with the present invention may be selected from the group consisting of inhibitors or activators of serotonin-mediated pathways, including therapeutics which are currently available, as well as those which are discovered, as described herein.
In accordance with the present invention, there is provided a method for the identification of a patient possessing a serotonin-mediated condition to be responsive to therapies for the condition, or to experience side-effects due to administration of a specific therapy. This method comprises:
(a) determining the characteristics of a serotonin-gated anion channel gene allele in a biological sample of the patient directly, using appropriate probes to a serotonin-gated anion channel, or indirectly, by phenotyping, and;
(b) correlating the genotype or phenotype with appropriated drug and/or dosage.
The presence or absence of a specific serotonin-gated anion channel allele indicates a predisposition, or lack thereof, to respond to serotonin-mediated therapies.
In another aspect, the invention claims a method for identifying a gene that is structurally related to a serotonin-gated anion channel. This method involves identifying a gene by designing probes or primers, including degenerate oligonulceotides, to specific sequences. These primers or probes encode structurally significant amino acid sequence (e.g., the sequence that forms the transmembrane portions of the serotonin-gated anion channel), and are used to screen large genomic or cDNA libraries. If a PCR approach is utilized, the primers are optionally designed to allow cloning of the amplified product into a suitable vector. PCR is particularly useful for screening cDNA libraries from rare tissue types. The method also includes identifying a gene using antibodies, using nucleic acid or amino acid scanning databases and computer programs, and screening for genes that function in a manner similar to, or different from, a serotonin-gated anion channel.
In a related aspect, the invention features the nucleic acid sequence identified by the method of identifying a gene which is structurally related to a serotonin-gated anion channel. This gene may be isolated from nematodes or mammals, preferably from rodents, and most preferably from human.
In another aspect, the invention features a transgenic, or other mutant animal, that over-expresses or under-expresses a serotonin-gated anion channel, or expresses a dominant-negative serotonin-gated anion channel. The invention includes a nematode or a non-human mammal, for example, a mouse, as the animal. In one embodiment, the animal over-expresses a serotonin-gated anion channel that is constitutively active.
In two additional aspects, the invention features a transgenic animal and methods of using the animal for the detection of therapeutics for conditions associated with serotonin-mediated cellular responses. Preferably the animal over-expresses a serotonin-mediated anion channel polypeptide, either wild-type or mutant, or expresses an antisense RNA to a serotonin-gated anion channel or a serotonin-gated anion channel fragment. In one embodiment, the animal also has a genetic predisposition to conditions associated with serotonin-mediated cellular responses.
In yet another aspect, the invention features a method for identifying a compound that modulates the activity of a serotonin-gated anion channel by exposing a nematode to a test compound, assaying the rate of locomotion, and comparing the locomotion rate to that of a nematode receiving no test compound, serotonin, or a placebo, where a modulation in the rate of locomotion indicates a compound that modulates the activity of a serotonin-gated anion channel. The test compound may be applied at various Concentrations. In addition, the nematodes used in the screen may be bacterial-lawn deprived prior to beginning the screen.
In still another aspect, the invention features a method for identifying a compound that modulates the activity of a serotonin-gated anion channel. The method involves exposing a nematode to test compound, quantifying the number of nematodes actively swimming after exposure to the compound, and comparing that number to that of a control receiving no test compound, serotonin, or a placebo, where a modulation in the number of actively swimming nematodes indicates a compound that modulates the activity of a serotonin-gated anion channel. The test compound may be applied at various concentrations. In addition, the nematodes used in the screen may be bacterial-lawn deprived prior to beginning the screen.
By xe2x80x9ctreatmentxe2x80x9d is meant the submission or subjection of an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell to a test compound or stimulus to a serotonin-mediated response.
By a xe2x80x9ctest compoundxe2x80x9d is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate serotonin-mediated cellular responses, by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
By xe2x80x9ctreatxe2x80x9d is meant to submit or subject an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell to a test compound or stimulus to a serotonin-mediated response.
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably the polypeptide is a serotonin-gated anion channel polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure serotonin-gated anion channel polypeptide may be obtained, for example, by extraction from a natural source (e.g., a neuronal or smooth muscle cell), by expression of a recombinant nucleic acid encoding a serotonin-gated anion channel polypeptide, or by chemically synthesizing the protein. Purity can be assayed by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, agarose gel electrophoresis, optical density, or HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
By a xe2x80x9cpurified nucleic acidxe2x80x9d is meant a nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By a xe2x80x9cserotonin-gated anion channelxe2x80x9d is meant a channel whose opening is regulated by serotonin binding to the channel. The opening of the channel selectively permits passage of anions from one side of the channel to the other. In one embodiment, the anion is chloride. Preferably the nucleic acid sequence encoding a serotonin-gated anion channel hybridizes to a mod-1 nucleic acid sequence.
By a xe2x80x9cmod-1 genexe2x80x9d is meant a gene encoding a polypeptide that is a serotonin-gated anion channel. In one embodiment, the mod-1 gene is from C. elegans. 
By a xe2x80x9cMOD-1 proteinxe2x80x9d or xe2x80x9cMOD-1 polypeptidexe2x80x9d is meant a polypeptide or fragment thereof, encoded by the mod-1 gene. In one embodiment, the MOD-1 protein or polypeptide is from C. elegans. 
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds to a serotonin-gated anion channel, but which does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample that naturally includes other proteins.
By xe2x80x9cmutantxe2x80x9d is meant different from what normally appears, occurs, or functions. As used herein, the term refers to a nucleic acid sequence that is different from the wild-type sequence. This term also describes a protein encoded by the mutant nucleic acid sequence. The term also means an organism that contains a mutant nucleic acid sequence.
By xe2x80x9cbiological activityxe2x80x9d is meant functional events mediated by a protein. In some embodiments, this includes events assayed by measuring the influx of ions into or out of a cell, or assaying the amount of serotonin binding to a channel. It also includes interactions of a polypeptide with another polypeptide. It also includes events that modify behavior or behavioral states. Such behavior includes, but is not limited to, movement, sexual behavior, or hallucinogenic behavior. Such behavioral states include, but are not limited to, migraine headaches, loss of appetite, gain of appetite, insomnia, inability to wake up, memory loss, nausea or vomiting, gastrointestinal cramps, ability or inability to learn, body temperature deregulation, moods, such as depression or mania, abnormal cardiovascular function, abnormal muscle contraction, and abnormal endocrine regulation.
As used herein, by xe2x80x9cmodulatesxe2x80x9d is meant increasing or decreasing the biological activity. Preferably the biological activity is increased or decreased 10% relative to a control. More preferably the biological activity is increased or decreased 50% relative to a control. Most preferably the biological activity is increased or decreased 90% relative to a control.
By xe2x80x9cassayingxe2x80x9d is meant analyzing the effect of a treatment or exposure, be it chemical or physical, administered to a whole animal or cells derived therefrom. The material being analyzed may be an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell. The analysis may be, for example, for the purpose of detecting current flow across a membrane, the rate of locomotion of an animal, altered gene expression, altered nucleic acid stability (e.g., mRNA stability), altered protein stability, altered protein levels, or altered protein biological activity. The means for analyzing may include, for example, recording current changes under voltage-clamp, voltage changes in current-clamp, or extracellular potentials, counting movements of an animal under a dissecting microscope, nucleic acid amplification techniques, reporter gene assays, antibody labeling, immunoprecipitation and phosphorylation assays, and other techniques known in the art for conducting the analyses of the invention.
By xe2x80x9cneuronxe2x80x9d is meant a cell of ectodermal embryonic origin derived from any part of the nervous system of an animal. Neurons express well-characterized neuron-specific markers that include neurofilament proteins, MAP2, and class III xcex2-tubulin. Included as neurons are, for example, hippocampal, cortical, midbrain dopaminergic, motor, sensory, sympathetic, septal cholinergic, and cerebellar neurons.
As used herein, by xe2x80x9cmeasuringxe2x80x9d is meant assessing an anion channel activity. Measuring can be done by use of standard electrophysiology voltage-clamping or patch-clamping equipment.
By xe2x80x9cconditionxe2x80x9d is meant a state of being or feeling. Conditions include, but are not limited to, migraine headaches, loss of appetite, gain of appetite, insomnia, inability to wake up, memory loss, nausea or vomiting, gastrointestinal cramps, ability or inability to learn, body temperature deregulation, moods such as depression or mania, abnormal sexual or hallucinogenic behavior, abnormal cardiovascular function, abnormal muscle contraction, and abnormal endocrine regulation.
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription of an operably-linked gene.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
By xe2x80x9cdominant-negativexe2x80x9d is meant a nucleic acid sequence encoding a polypeptide which when expressed acts in a way to inhibit another polypeptide. This term also refers to the polypeptide itself. In one embodiment, the polypeptide that is inhibited is a wild-type polypeptide, and the dominant negative sequence encodes a mutant polypeptide of the same gene.
As referred to herein, by xe2x80x9cconstitutively activexe2x80x9d is meant a nucleic acid sequence that encodes a polypeptide, which when expressed is in an active form at least as, or more often as the wild-type polypeptide is, in a cell in which wild-type polypeptide is naturally expressed. The polypeptide may be in an active form by being phosphorylated, or dephosphorylated, or cleaved from a propeptide to a peptide, or being ligand independent, or being mutated.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell or organism that includes a DNA sequence (transgene) that is inserted by artifice into a cell and becomes part of the genome of the organism that develops from that cell. As used herein, the transgenic organism is generally a transgenic non-human mammal (e.g., rodents such as rats or mice) or invertebrate (e.g., Caenorhabditis elegans).
By xe2x80x9cantisensexe2x80x9d is meant a nucleic acid sequence, regardless of length, that is complementary to the coding strand gene encoding a serotonin-gated anion channel. Preferably the antisense nucleic acid is capable of decreasing the activity of a serotonin-gated anion channel when present in a cell that normally is modulated by serotonin. Preferably the decrease is it least 10%, relative to a control, more preferably 25%, and most preferably 95%.
By xe2x80x9cexposexe2x80x9d is meant to allow contact between an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell, and a test compound or activator of a serotonergic response.
By xe2x80x9ccharacteristicsxe2x80x9d is meant properties or features. Characteristics include, but are not limited to, the nucleic acid sequence of a gene, or various alleles of a gene, the amino acid sequence of a protein, the level of expression of proteins or mRNA, and altered protein levels in tissues.
By xe2x80x9cinhibitxe2x80x9d is meant to decrease the level of expression of a serotonin-gated anion channel, or to decrease the function or activity of a serotonin-gated anion channel. Preferably the expression, function, or activity of the channel is deceased at least 25%, more preferably at least 50%, 70%, or 80%, and most preferably at least 95%, compared to a control (e.g., one which is not contacted with a test compound or an antisense nucleic acid).